Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR

Initial screening of the expression pattern demonstrated that 1 of the 7 genes was expressed at very low levels in reticulocytes and was excluded from further analysis. The range of expression stability of the other 6 genes was (from most stable to least stable): GAPDH (glyceraldehyde 3-phosphate dehydrogenase), SDHA (succinate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyl transferase 1), HBS1L (HBS1-like protein) and AHSP (alpha haemoglobin stabilising protein), followed by B2M (beta-2-microglobulin).Using this simple approach, GAPDH was found to be the most suitable housekeeping gene for expression studies in reticulocytes while the commonly used B2M should be avoided.Reticulocytes are juvenile enucleated red cells produced during erythropoiesis and spend approximately 24 hours in the bone marrow before entering the peripheral circulation. Reticulocytes persist for a few days in the circulation before forming the slightly smaller, mature red blood cell at which time any residual RNA the reticulocytes still possessed, is lost [1]. Peripheral blood reticulocytes are not only readily accessible, but remnants of RNA they still harbour are likely to represent gene expression profiles of patients. This makes them potentially very useful in looking for subtle changes in gene expression, particularly for quantitative traits involved in the phenotypic outcome of haemoglobinopathies. In order to reach the sensitivity needed to detect such subtle changes in gene expression, quantitative real time PCR (Q-RT-PCR) was employed.When comparing gene expression in different samples it is crucial to consider experimental variations such as amount of starting material, RNA extraction and reverse transcription efficiencies. To account for these, accuracy of Q-RT-PCR relies on normalisation to an internal control, often referred to as a housekeeping gene [2,3]The prerequisite of a suitable housekeeping gene is that it should, of course, be adequately expressed in the tissue of int